The Notch pathway is a conserved signaling mechanism that functions to modulate cell-fate decisions. The overall objectives of this proposal are to define roles for Notch in cell-fate determination during vascular development, physiological angiogenesis and maintenance of vasculature. Our general hypothesis is that Notch is critical for arterial-venous, venous-lymphatic development and for physiological angiogenesis. Our general strategy will use a combination of in vitro angiogenesis/lymphangiogenesis assays and mouse modeling to define the consequences of altering Notch activity in endothelial cells. Our preliminary studies using in vitro assays suggest that Notch promotes sprouting and survival of blood endothelial cells but blocks capillary-like cord formation. In Aim I, using in vitro angiogenesis assays, we evaluate signaling mechanisms that function downstream of Notch, with a focus on the PI-3K/Akt and VEGF-C/VEGFR-3 pathways. In Aim II, we evaluate the VEGFR-3 gene as a direct transcriptional target of Notch and explore a role for Notch in lymphangiogenesis by manipulating cultured lymphatic endothelial cells. In Aim III, we use mouse models that activate or inactivate Notch in embryonic vasculature to examine Notch function. We have developed mouse models that conditionally activate Notch signaling in the vasculature to define Notch action at various stages of embryogenesis. Notch activation in embryonic vasculature will be used to evaluate roles for Notch in arterial/venous and venous/lymphatic specification, recruitment of vascular smooth muscle cells, vascular remodeling, and heart development. Notch genes are expressed in adult vasculature but their postnatal function is unknown. In Aim IV, Notch will be conditionally activated in adult vasculature to evaluate function in vascular integrity. In vivo assays will examine whether Notch contributes to, or interferes, with VEGF- or FGF-induced angiogenesis. Finally, Notch function will be studied in the ovary, which has robust physiological angiogenesis during folliculogenesis and corpus luteum formation. Using a hypophysectomized mouse model that allows hormonal induction of ovarian angiogenesis, we will probe Notch function in ovarian vasculature. These mice will be used to define the consequences to ovarian angiogensis when Notch is conditionally activated in ovarian vessels or interfered with using Notch antagonists. Our long-term objective is to understand Notch function in human vascular disorders and during pathological angiogenesis.